The objective of the research proposal is to use the recently cloned mouse polymerase gamma in characterizing the biochemistry of this enzyme and in generating mutant versions of the protein which are defective in the proofreading 3/-5/ exonuclease activity. There are four Specific Aims of this proposal: 1. Confirm the mitochondrial location and function of the putative catalytic subunit of the mouse mtDNA polymerase. 2. Purification and enzymatic characterization of the mouse mtDNA polymerase. 3. Mutagenesis of the 3/-5' exonuclease domains of the mouse mitochondrial DNA polymerase. 4. Generation of transgenic mice carrying the 3'-5' exonuclease deficient mutator version of the mtDNA polymerase. This proposal is related to health issues which focus on the bioenergetic needs of all cells. Maintenance of the integrity of the mitochondrial genome is essential for mitochondrial function. Much attention has recently been paid to various encephalomyopathies, antiviral treatments using nucleoside analogs such as AZT, and in the aging process where mutations and deletions of mtDNA are consequential. This work will significantly increase our understanding of mammalian mitochondrial DNA replication and maintenance of the essential mitochondrial functions.